Figure S2. Regulation of mRNA association with polysomes in response to low oxygen stress and reoxygenation treatments. Venn diagram of genes with significant changes (FDR cut off = 0.001) in immunopurified mRNA levels, (a) Increase in response to 2 h (2HS) or 9 h (9HS) and decrease after 1 h of reoxygenation (1R) and (b) Decrease in response to HS and increase after 1 h of reoxygenation. mRNAs detected with a Present call in all total mRNA samples and showing call changes in the IP fraction (as predicted by mas 5.0) as a result of 2HS and 9HS are represented in panels (c) All Present to All Absent calls and (d) All Absent to All Present call.

Figure S3. Multivariate statistical evaluation of 1H NMR spectra of metabolites from control and low oxygen stressed seedlings. In the PCA plots samples are represented with individual symbols with the treatments color-coded as follows, 2NS(&#UF075;), 9NS (&#UF0A2;), 2HS (&#UF0C5;), 9HS (&#UF0B3;), and 1R (&#UF072;). (a) PCA scores plot for the first two principal components. (b) Loadings for the scores plot in (a). (c) PCA scores plot using only the 12 most significant integral regions (variables) determined from the loadings plot in (b). The PCA model was cross-validated by alternately including and omitting the 12 most significant spectral regions (Figure S3c, d). In (c) the sample groupings observed in (a) are retained, although they appear in a different order. (d) PCA scores plot for all of the integral regions except for the 12 most significant regions in (b). (e) Explained variance plot. Principal component (PC) 1 and 2 explain more than 67% of the variance in the data, whereas PC3 primarily describes the biological variance in sugars.

Table S1. Evaluation of reproducibility between biological replicates. Total and Polysomal mRNA samples extracted from Arabidopsis 7-day-old seedlings were prepared from three independent biological replicates and hybridized to Affymetrix ATH1 chips. Reproducibility between replicates was evaluated by Pearson correlation of microarray data obtained and is expressed in R2 values.

Expression data obtained in CEL files was extracted by use of the Bioconductor package of the R statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA. (b) Detection call of all genes on the ATH1 GeneChip. Expression data obtained in CEL files was extracted by use of the Bioconductor package of the R statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 Signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA. (c) Signal obtained for all genes on the ATH1 GeneChip. Expression data obtained in CEL files was extracted by use of the Bioconductor package of the r statistical software and normalized using Robust Multichip Average (RMA). The signal data obtained from the RMA analyses of the immunopurified polysomal RNA was further adjusted to account for variation in global polysome level under the different treatment conditions (normalization factors used for polysomal RNA samples were 2NS, 0.7485; 9NS, 0.7463; 2HS, 0.4253; 9HS, 0.3753; 1R, 0.6723). Data presented are the log2 Signal values obtained for each probe-pair from each replicate hybridization onto Affymetrix ATH1 chips. The area shaded in gray includes comparisons between treatments expressed as signal log2 ratio (SLR) and FDR cut-offs. The area shaded in blue provides the mas 5.0 detection calls for each gene on each chip. 2NS, 2 h non-stress; 2HS, 2 h hypoxia-stress; 9NS, 9 h non-stress; 9HS, 9 h hypoxia stress; 1R, 1 h reoxygenation after 9 h hypoxia-stress; T, Total mRNA; IP, Immunopurified polysomal mRNA.

Table S3. Relative porportion of mRNA present in each group of genes when compared to total RNA detected for the whole data set (Present Calls). Relative proportion of mRNA present in each cluster when compared to total RNA detected for the whole data set (Present Calls): An estimate of the proportion of cellular mRNA represented in each cluster was obtained by calculating the sum of signal data, a measure of the amount of RNA detected, as compared to the sum of the signal data of the whole set of genes (n = 8863 genes). Values are percent mRNA of Total mRNA in the data set. RPs, ribosomal proteins (n = 174 probe pairs).

Table S4. Functional categorization of genes with increased association with polysomes in response to HS. Transcripts detected as Present in all 30 chips and showing significant increase in IP RNA levels (FDR cutoff = 0.001) exclusively after 2 h or after 9 h HS were compared and evaluated for the predicted biological process for the encoded gene product. This analysis was performed using the TAIR gene ontology retrieval tool http://www.arabidopsis.org/tools/bulk/index.jsp.

Table S5 (b) Annotation and functional category of gene cluster members, as designated by the MapMan software. Some loci are annotated in more than one functional category. A statisitical analysis of representation of categories for each cluster is presented in the sheets labelled Cluster 1 to 10.

Table S6. Dynamics in mRNA accumulation and translation of mRNAs encoding proteins with related function or processes. Data in this table are SLR data used for the heat map displays in Figure 4b, A2.

Table S7. Quantification of metabolites measured by coupled enzyme spectrophotometricand assay. Pulverized seedling tissue was extracted in perchloric acid, neutralized and an aliquot used to assay specific metabolites in the presence of the metabolite specific converting enzyme and co-factor (NAD+ or NADH) and reactions were driven to completion. Values presented are absolute concentrations (average of 3 or more bioreplicates) and standard deviation for the quatifiable metabolites. This data was used to calculate and evaluate statistical significance of differences between treatments (fold change). Lactate values for 2NS and 9NS samples may be underestimates because of low content in non-stressed samples.

Appendix S1. Supplemental experimental procedures.

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.